This proposal seeks to identify the exact step(s) of adenovirus (Ad) infection that is (are) responsible for the initiation of an anti-Ad acute inflammatory response upon systemic virus application. Over the last two decades numerous Ad-based vectors have been developed for gene therapy applications and many are currently being tested in clinical trials. Most recently, interest in Ad has further expanded due to its potential as a vector for vaccination against life threatening infectious agents such as anthrax. While natural infections with Ad are largely harmless to humans, intravenous Ad administration may result in a severe inflammatory response, which can lead to fatal outcomes. It is currently recognized that the initiation of this acute systemic inflammation depends on interactions of the Ad capsid with host cells. Despite significant knowledge regarding Ad interactions with cells in vitro, the molecular mechanisms governing Ad bio-distribution, hepatic tropism and toxicity in vivo remain poorly understood. Recently, we identified a novel blood factor-dependent pathway of Ad liver cell infection in vivo. This finding explained the observed Ad biodistribution in animals after systemic application and is fundamental for the development of novel strategies to modify both Ad tropism in vivo and virus-associated toxicity. In mouse models, we will analyze the innate immune response to intravenously applied capsid-modified Ad mutants deficient in their ability to undergo the initial steps of virus infection. Our specific aims are to analyze the role of Ad interactions 1) with primary attachment receptors;2) with integrins, which facilitate Ad initialization;or 3) with cellular factors upon virus escape from endosomes, in the initiation of an anti-Ad inflammatory response. Based on the data obtained, in our fourth specific aim we will construct ah Ad vector ablated for binding to known virus receptors and capable of infecting cells via an alternative receptor, LDLR. By applying this vector in mice and evaluating its toxicity we will test the hypothesis that modification of the Ad capsid represents a feasible approach to reduce acute Ad-mediated inflammation while preserving virus gene transfer ability upon systemic application. These studies will significantly improve our understanding of fundamental mechanisms of the host defense against viral pathogens and may ultimately lead to the development of safe and efficient Ad vectors for the therapy of a wide range of inborn and acquired human diseases.